Corporate Brand Building in Start-Ups through Equity-Based Crowdfunding: A Case Study of Virtuous Spirits

This study investigates if and how equity-based crowdfunding can benefit a start-up’s corporate brand building process. Building the corporate brand can play a significant role for the company’s establishment in the market. Although equity-based crowdfunding has grown exponentially in the Nordics, it has not been covered enough in corporate brand building literature. The scope of this research was to shed light on the yet relatively undiscovered area of equity-based crowdfunding as a corporate brand building tool. A case study approach was chosen in order to produce in-depth information about a start-up’s corporate brand building process. Accordingly, ten semi-structured in-depth interviews with the start-up Virtuous Spirits’ creators, investors, non-investors and the intermediary of the equity-based crowdfunding campaigns were conducted. The results suggest that through equity-based crowdfunding, the start-up can find the right brand ambassadors leading to positive word-of-mouth and feedback which is crucial for its corporate brand building. This is possible due to the pre-targeted audience provided by the intermediary. The importance of the intermediary is further highlighted through its positive effect on the start-up’s credibility. The study therefore concludes in the proposition to use equity-based crowdfunding as a channel of corporate communication, aligning the corporate image with the corporate identity in order to build the start-up’s corporate brand

Thiol-gelatin-norbornene bioink for laser‐based high‐definition bioprinting

Two-photon polymerization (2PP) is a lithography-based 3D printing method allowing the fabrication of 3D structures with sub-micrometer resolution. This work focuses on the characterization of gelatin-norbornene (Gel-NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol-ene system allows 2PP processing of cell-containing materials at remarkably high scanning speeds (1000 mm s(-1)) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2-0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel-NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki-67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high-definition bioprinting of well-defined constructs for long-term cell culture studies

On-chip high-definition bioprinting of microvascular structures

'Organ-on-chip' devices which integrate three-dimensional (3D) cell culture techniques with microfluidic approaches have the capacity to overcome the limitations of classical 2D platforms. Although several different strategies have been developed to improve the angiogenesis within hydrogels, one of the main challenges in tissue engineering remains the lack of vascularization in the fabricated 3D models. The present work focuses on the high-definition (HD) bioprinting of microvascular structures directly on-chip using two-photon polymerization (2PP). 2PP is a nonlinear process, where the near-infrared laser irradiation will only lead to the polymerization of a very small volume pixel (voxel), allowing the fabrication of channels in the microvascular range (10-30 mu m in diameter). Additionally, 2PP not only enables the fabrication of sub-micrometer resolution scaffolds but also allows the direct embedding of cells within the produced structure. The accuracy of the 2PP printing parameters were optimized in order to achieve high-throughput and HD production of microfluidic vessel-on-chip platforms. The spherical aberrations stemming from the refractive index mismatch and the focusing depth inside the sample were simulated and the effect of the voxel compensation as well as different printing modes were demonstrated. Different layer spacings and their dependency on the applied laser power were compared both in terms of accuracy and required printing time resulting in a 10-fold decrease in structuring time while yielding well-defined channels of small diameters. Finally, the capacity of 2PP to create vascular structures within a microfluidic chip was tested with two different settings, by direct embedding of a co-culture of endothelial- and supporting cells during the printing process and by creating a supporting, cell-containing vascular scaffold barrier where the endothelial cell spheroids can be seeded afterwards. The functionality of the formed vessels was demonstrated with immunostaining of vascular endothelial cadherin (VE-Cadherin) endothelial adhesion molecules in both static and perfused culture

Impact of hydrogel stiffness on differentiation of human adipose-derived stem cell microspheroids

Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3- to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE. Impact Statement Osteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE

Cross-linkable gelatins with superior mechanical properties through carboxylic acid modification : increasing the two-photon polymerization potential

The present work reports on the development of photo-cross-linkable gelatins sufficiently versatile to overcome current biopolymer two-photon polymerization (2PP) processing limitations. To this end, both the primary amines as well as the carboxylic acids of gelatin type B were functionalized with photo-cross-linkable moieties (up to 1 mmol/g) resulting in superior and tunable mechanical properties (G' from 5000 to 147000 Pa) enabling efficient 2PP processing. The materials were characterized in depth prior to and after photoinduced cross-linking using fully functionalized gelatin-methacrylamide (gel-MOD) as a benchmark to assess the effect of functionalization on the protein properties, cross-linking efficiency, and mechanical properties. In addition, preliminary experiments on hydrogel films indicated excellent in vitro biocompatibility (close to 100% viability) both in the presence of MC3T3 preosteoblasts and L929 fibroblasts. Moreover, 2PP processing of the novel derivative was superior in terms of applied laser power (>= 40 vs >= 60 mW for gel-MOD at 100 mm/s) as well as post-production swelling (0-20% vs 75-100% for gel-MOD) compared to those of gel-MOD. The reported novel gelatin derivative (gel-MOD-AEMA) proves to be extremely suitable for direct laser writing as both superior mimicry of the applied computer-aided design (CAD) was obtained while maintaining the desired cellular interactivity of the biopolymer. It can be anticipated that the present work will also be applicable to alternative biopolymers mimicking the extra cellular environment such as collagen, elastin, and glycosaminoglycans, thereby expanding current material-related processing limitations in the tissue engineering field

Photo-crosslinkable recombinant collagen mimics for tissue engineering applications

Gelatin is frequently used in various biomedical applications. However, gelatin is generally extracted from an animal source, which can result in issues with reproducibility as well as pathogen transmittance. Therefore, we have investigated the potential of a recombinant peptide based on collagen I (RCPhC1) for tissue engineering applications and more specifically for adipose tissue regeneration. In the current paper, RCPhC1 was functionalized with photo-crosslinkable methacrylamide moieties to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The resulting biomaterial (RCPhC1-MA) was characterized by evaluating the crosslinking behaviour, the mechanical properties, the gel fraction, the swelling properties and the biocompatibility. The obtained results were compared with the data obtained for methacrylamide-modified gelatin (Gel-MA). The results indicated that the properties of RCPhC1-MA networks are comparable to those of animal-derived Gel-MA. RCPhC1-MA is thus an attractive synthetic alternative for animal-derived Gel-MA and is envisioned to be applicable for a wide range of tissue engineering purposes

High-resolution 3D bioprinting of photo-cross-linkable recombinant collagen to serve tissue engineering applications

Various biopolymers, including gelatin, have already been applied to serve a plethora of tissue engineering purposes. However, substantial concerns have arisen related to the safety and the reproducibility of these materials due to their animal origin and the risk associated with pathogen transmission as well as batch-to-batch variations. Therefore, researchers have been focusing their attention toward recombinant materials that can be produced in a laboratory with full reproducibility and can be designed according to specific needs (e.g., by introducing additional RGD sequences). In the present study, a recombinant protein based on collagen type I (RCPhC1) was functionalized with photo-cross-linkable methacrylamide (RCPhC1-MA), norbornene (RCPhC1-NB), or thiol (RCPhC1-SH) functionalities to enable high-resolution 3D printing via two-photon polymerization (2PP). The results indicated a clear difference in 2PP processing capabilities between the chain-growth-polymerized RCPhC1-MA and the step-growth-polymerized RCPhC1-NB/SH. More specifically, reduced swelling-related deformations resulting in a superior CAD-CAM mimicry were obtained for the RCPhC1-NB/SH hydrogels. In addition, RCPhC1-NB/SH allowed the processing of the material in the presence of adipose tissue-derived stem cells that survived the encapsulation process and also were able to proliferate when embedded in the printed structures. As a consequence, it is the first time that successful HD bioprinting with cell encapsulation is reported for recombinant hydrogel bioinks. Therefore, these results can be a stepping stone toward various tissue engineering applications

Laser-based 3D printing of hydrogel barrier models for microfludic applications

The placenta secures the survival and development of the fetus. As placental tissue connects the fetus with the mother and is responsible for endogenous and exogenous material transfer. The maternal and fetal blood are thereby separated, by the so-called placental barrier, which is made up by the trophoblastic syncytium and the fetal capillary wall. Research in the field of placenta biology represents a challenging topic, as current approaches are difficult to perform, time consuming and often carry the risk of harming the fetus. The establishment of a reproducible in-vitro model, simulating the placental transport is necessary to study fetal development and for identification of underlying causes of maldevelopment. In this study, a photosensitive hydrogel material, in combination with two-photon polymerisation, was used to produce high resolution structures with nanometre precision geometries. Gelatine modified with methacrylamide and amino-ethyl-methacrylate (GelMOD AEMA) was thereby crosslinked within a customised microfluidic-device under the addition of photoinitiator, separating the chip in two different compartments (Figure 1). The fetal compartment contains HUVEC cells which are cultivated in EGM2, while BeWo B30 cells are supplied with DMEM Ham-F12 to mimic the maternal compartment. This microfluidic approach in combination with native flow profiles can be used to precisely remodel the microenvironment of placental tissue. The establishment of a functional placenta-on-a-chip-model allows the modulation of different clinical and biological scenarios in the future. A potential application can be found in the simulation of altered sugar transport across the placental membrane and evaluation of the effects of altered nutrient balance in-utero Please click Additional Files below to see the full abstract

Thiol-norbornene gelatin hydrogels : influence of thiolated crosslinker on network properties and high definition 3D printing

Photocrosslinkable gelatin hydrogels are excellent bioinks or biomaterial ink components to serve biofabrication applications. Especially the widely investigated gelatin-methacroyl (gel-MA) hydrogels hold an impressive track record. However, over the past decade, increasing attention is being paid to thiol-ene photo-click chemistry to obtain hydrogel networks benefitting from a faster reactivity (i.e. seconds vs minutes) along with superior biocompatibility and processability. In order to exploit this photo-click chemistry, often an ene-functionality (e.g. norbornene) is introduced onto gelatin followed by crosslinking in the presence of a multifunctional thiol (e.g. dithiothreitol). To date, very limited research has been performed on the influence of the applied thiolated crosslinker on the final hydrogel properties. Therefore, the present work assesses the influence of different thiolated crosslinkers on the crosslinking kinetics, mechanical properties and biological performance of the hydrogels upon encapsulation of primary adipose tissue-derived stem cells which indicated a cell viability exceeding 70%. Furthermore, the different formulations were processed using two-photon polymerization which indicated, in addition to differences in processing window and swelling ratio, a previously unreported phenomenon. At high intensities (i.e. 150 mW), the laser results in cleavage of the gelatin backbone even in the absence of distinct photo-cleavable functionalities. This can have potential to introduce channels or softer regions in gels to result in zones characterized by different degradation speeds or the formation of blood vessels. Consequently, the present study can be used to provide guidance towards tailoring the thiol-ene system towards the desired applications